The studies proposed in this application continue efforts to elucidate von Willebrand factor (VWF) in the VWF A1 domain (VWFA1) can differentially regulate specific adhesive functions. Experimental evidence indicates that the interaction of glycoprotein (GP) Ib1 with VWFA1 immobilized onto a surface, leading to platelet adhesion, or in solution, mediating platelet aggregation, may occur through different mechanisms. Our goal is to define the distinctive structural determinants underlying the initiation and regulation of these diverse VWF activities and establish their functional relevance in models of vascular injury. In aim 2 we intend to ascertain whether 1-thrombin is a physiologic modulator of VWF adhesive properties. We hypothesize that VWFA1 and 1-thrombin establish inter- molecular contacts when bound to the same GPIb1 receptor, and this influences the stability of the VWFA1-GPIb1 bond independently of shear stress. We propose to identify the VWFA1 residues that support the interaction with GPIb1-bound 1-thrombin, thus defining a novel mechanism for the regulation of VWF function during thrombogenesis. In aim 3 we propose to evaluate whether a specific IgG found in the human population is a modulator of VWF activity. We have identified in human and mouse blood a specific IgG that binds selectively to VWFA1, and obtained preliminary evidence that this IgG may play a role in thrombus formation. Our goal is to characterize the structure of the specific IgG, define the mode of interaction with VWFA1 and obtain definitive in vivo evidence for its physiopathological significance. In aim 4 we intend to define the signaling function of collagen- bound VWF leading to platelet activation. We have characterized the distinctive intracytoplasmic Ca++ signals that follow platelet adhesion to immobilized VWFA1 or collagen under flow conditions, and found that they are enhanced when platelets interact with collagen-bound VWF. We propose to dissect the mechanisms of platelet activation supported by the collagen-VWF complex and the effects of hydrodynamic force on the process. The results of this research will improve our ability to influence disease processes that involve platelets in atherothrombosis. PUBLIC HEALTH RELEVANCE: Experimental and clinical evidence points to a key role of VWF in thrombus formation, orchestrated by interactions with other proteins and modulated through forces generated by flowing blood. Understanding these different functions requires addressing structural details as well as verification of concepts in intravidal models. This will lead to a better diagnosis and treatment for cardio- and cerebro-vascular diseases.